Effects of Calendula Officinalis and Hypericum Perforatum on Antioxidant, Anti-Inflammatory, and Histopathology Indices of Induced Periodontitis in Male Rats

Statement of the Problem: Periodontitis is one of the most common bacterial infections of the oral cavity. It is important to find adjunctive methods for chemical treatment of periodontitis with less complications and proven therapeutic properties. Purpose: The aim of this study was to compare the effects of Calendula officinalis and Hypericum perforatum on antioxidant, anti-inflammatory and histopathologic indices of induced periodontitis in male rats. Materials and Method: In this experimental animal study, forty adult male Sprague-Dawley rats were randomly divided into 4 groups (n=10) and then experimental periodontitis was induced by 3-0 nylon non-absorbable ligature. Each group was treated for 10 days as follows: 1) H. perforatum hydroalcoholic extract, 1000 mg/kg/daily, orally; 2) C. officinalis hydroalcoholic extract, 1000 mg/kg/daily, orally; 3) a mix of the two plants, 1000 mg/kg/ daily, orally, 4) normal saline solution. At the end of study, blood sample were obtained via cardiocentesis, the rats were euthanized, and their maxillae were removed. The samples were analyzed for histopathological scores and total antioxidant capacity and IL-1β were measured. Results: Mixed hydroalchoholic extract of H. perforatum and C. officinalis decreased IL-1β (4.3020±0.63), and increased the antioxidant parameter in comparison to the control group (3.1192±0.43) (p< 0.001). There were significant histopathological differences between the treatment groups and the control group. Conclusion: Mixed hydroalchoholic extract of H. perforatum and C. officinalis might be considered as an adjunctive treatment for periodontitis.


Introduction
Periodontal disease is a major public health problem worldwide. It is a pathological condition with detrimental effects on the toot-supporting structures and tissues and is the most important cause of tooth loss in the adult population [1]. Inflammatory cells, especially polymorphonuclear cells, release free radicals of oxygen. Therefore, periodontitis occurs because of a decrease in antioxidant capacity and increased oxidative damage. The host's inflammatory cells release several factors, which can lead to bone loss in vitro. These factors include prostaglandins, interleukin 1-α and 1-β, and Effect of Calendula Officinalis and Hypericum Perforatum on Induced Periodontitis Tanideh N, et al. 10.30476/DENTJODS.2020.83660.1056 315 tumor necrosis factor-α (TNF-α) [2].
Pain, discomfort, cosmetic problems, and tooth loss are some of the disorders associated with periodontitis.
The goal of periodontal treatment is to reduce inflammation in the inflamed tissue, decrease the counts of pathogenic bacteria, and eliminate periodontal pockets.
Mechanical therapy, antimicrobial drugs, and use of systemic antibiotics are some of the clinical techniques used in the treatment of periodontitis [3].
Scaling and root planing are one of the most commonly used and effective mechanical interventions for periodontitis, which result in a decrease in pocket depth, inflammation severity and an improvement in the attachment level. While mechanical treatment including scaling and surgery can eliminate microorganisms and are the main treatment way, antibiotic therapy is also considered as an adjunctive treatment. However, systemic antibiotics might be associated with complications, including drug interactions and organ damage [4].
Herbal plants have been reported to have an important role in the treatment of various diseases. As a result, it might be of great interest to use herbal medicines as an alternative to synthetic antibiotics, in association with mechanical removal of debris [5].
Calendula officinalis (C. officinalis), also known as Marigold, is an important medicinal plant in the Asteraceae family. In traditional medicine, it is used to treat fever and cancer [6] due to its antioxidant and antiinflammatory compounds [7]. The plant is rich in various pharmaceutically active ingredients, including sterols, flavonoids, carotenoids, and glycosides [8]. Over 100 different combinations of C. officinalis are extracted. Quercetin, as the main ingredient in this plant, is responsible for the plant's major anti-inflammatory and antioxidant effects [9].
Hypericum perforatum (H. perforatum), known as St. John's wort, is an herbal medicinal plant from the family of Clusiaceae. [10]. It exhibits antibacterial and antiviral effects through partial control of the transcription factor NF-κB, and by involving some serine/ threonine kinases from the protein kinase C (PKC) family.
The main ingredients responsible for pain relief in H.
perforatum are hyperforin and hypericin.The main antiinflammatory and antioxidant properties of this plant are attributed to its diverse components, including hypersin and pseudo-pyrcine and flavonoids, such as quercetin [11][12][13]. Considering the ever-increasing administration of chemical drugs/agents and their side effects, including anaphylactic reactions, opportunistic infections and bacterial resistances to commonly used antibiotics, and also because of the popularity of herbs due to their lower cost and lower complications [14], we decided to evaluate C. officinalis and H. perforatum plants with anti-inflammatory, antioxidant and antimicrobial properties as an adjunctive treatment for mechanical and chemical therapy in periodontitis.

Materials and Method
The experiments were carried out in accordance with h and stored at -20°C. Finally, the extracts were con-verted to a solution of 100 mg/kg to be used for daily consumption of rats [15]. Plants species were determined in the Department of Botany of Shiraz University of Medical Sciences.

Induction of periodontitis
The rats underwent anesthesia with 10% Ketamine hydrochloride (90 mg/kg, IM) and 2% Xylazine (5 mg/kg, IM) and nylon 3-0 non absorbable ligature (ETHIBOND EXCEL) polyester green coated braided were wrapped up around the second maxillary molar tooth of the left side and tied to the palatal area in order to induce periodontitis ( Figure 1). The rats were randomly divided into 4 groups of 10, to demonstrate induction of experimental periodontitis [16]. were fed daily for 10 days by gavage method and the rats were euthanized ethically on day 11.
-Group 4: One mL of normal saline solution was fed orally daily for 10 days by gavage method and the rats were euthanized ethically on day 11. Animals were observed until the 11th day, the period of the most intense alveolar bone loss [16][17]. All the rats were euthanized ethically by 70% CO 2 .
Blood was prepared via cardiocentesis and serum was transferred to laboratory for inflammatory and antioxidant capacity tests. The whole maxilla was harvested and soft tissues were separated from bone and placed in 10% formalin for histopathological evaluation.

picrylhydrazyl (DPPH) assay
The initial concentration of reagents was selected based on their absorbance close to 1.0 at measured wavelengths. As a result, freshly prepared methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with a HCl, and 20 mM FeCl 3 ·6H 2 O solution was prepared freshly and warmed at 37°C. Aliquots of 20μL of the plant extract diluted in methanol (at different concentrations) were mixed with 180μL of FRAP reagent [19].
The absorbance of reaction mixture was measured at 595nm after 10 minutes.

IL1-β Test
Five mL of blood was taken from the rats. After centrifuging at 5000r/min for 5min, the supernatant was obtained and stored at -80°C. An eBioscience kit (eBioscience, San Diego, CA) was purchased and the serum level of inflammatory factor interleukin-1β (IL-1β) was determined by enzyme linked immunosorbent assay (ELISA) [20].

Histopathological assessments
The tissue obtained from the left side of the maxilla was placed in 10% formalin and prepared for histopatholog-ical examination in the standard fashion; the sections were stained with hematoxylin-eosin (H&E) staining.
The H&E slides were investigated under a light microscope carefully and different magnifications in a blind manner.

Investigation of inflammation and alveolar bone loss
The area between the first and second molar teeth was studied at ×40 magnification. The parameters, such as infiltration of inflammatory cells, alveolar bone integrity and cementum and collagen degradation were evaluated and graded ranging from minor infiltration of inflammatory cells and no collagen degradation to severe infiltration of inflammatory cells and collagen degradation scored from 0-3 respectively. These scores were defined as score 0: absence of or only discrete cellular infiltration, few osteoclasts, preserved alveolar process and cementum; score 1: moderate cellular infiltration, presence of some osteoclasts, some but minor alveolar process resorption and intact cementum; score 2: accentuated cellular infiltration, large number of osteoclasts, accentuated degradation of the alveolar process and partial destruction of cementum; and score 3: accentuated cellular infiltrate and total destruction of alveolar process and cementum [21].

Statistical analysis
After assessing the normal distribution of the data with the Kolmogorov-Smirnov test, one-way ANOVA was used to make comparisons between the groups. The nonparametric equivalence-Kruskal-Wallis test was used for quantitative data. To compare qualitative factors such as histopathological scores between the different groups on different days, Mann-Whitney U test was used.
A p value of ≤0.05 was considered statistically significant. All the statistical analyses were carried out by SPSS20.

IL-1B Test
The results of this test were presented based on the serum levels of IL-1 beta (pg/mL) ( Table 2). There were significant differences between the experimental groups and the normal saline group (p<0.05).   The lowest and highest levels were recorded in the mixed (mean= 4.302±0.62) and the normal saline groups (mean= 9.164±1.34), respectively (Figure 3).
In the C. officinalis group, 70% were mild and 30% were moderate. In the H. perforatum group, 50% were mild and 50% were moderate. In the combination group, 80% were mild and 20% were moderate.  In the normal saline group, 20% were moderate and 80% were severe. The highest collagen degradation and an interdental abscess in the first and second molar areas were observed in the normal saline group, and the lowest was seen in the combined group ( Figure 5).

Discussion
The results of the present study showed that use of a mixture of hydroalcoholic extracts of C. officinalis and H. perforatum for the treatment of periodontal disease resulted in a decrease in the degree of inflammation, alveolar bone loss, and oxidative stress of the tissue. IL-1β is a multifunctional cytokine and plays an active role in inflammation, immunity, and bone metabolism. Evidence shows that it promotes bone resorption, and its level is significantly correlated with periodontal attachment loss [22].
Alexandre et al. [11] showed that C. officinalis decreased the bone loss as well as the level of inflammatory mediators such as IL-1βon experimental periodontitis in rats. It was shown that the strong anti-inflammatory Tanideh et al. [14] reported that acceleration of the healing process of mucosa in male rats treated with C.
officinalis was significantly better than that in the control group. According to Preethi et al. [24], the phyto- The non-enzymatic system, such as ascorbic acid, phenolics, and flavonoids as natural phyto-antioxidants, has a vital role in the development, cellular protection, and defense response against oxidative stress [26].
DPPH and FRAP are regarded as the most acceptable method to determine in vitro the antioxidant potential of the plant samples, which was one of the strengths of our study.
Collagen is the main constituent of periodontal ligament, with a key role in the architecture of periodontium. De Almeida et al. [27] showed that collagen breakdown is the main marker of the progression of periodontal disease. They verified that C. officinalis extract decreased collagen breakdown and increased collagen concentration. [27] In the present study, the lowest grade of collagen degradation was observed in the group treated with C. officinalis and the combined group compared to normal saline solution group.
Heijnen et al. [28] showed that C. officinalis decreased periodontal oxidative stress, which was attributed to the presence of two antioxidant pharmacophores within the quercetin molecule that have the optimal configuration for free radical scavenging. There was a positive correlation between phenolic compounds and antioxidant activity of C. officinalis and C. officinalis is rich in polyphenolic compounds.
Histopathologic and radiographic evaluation of myoperoxidase, P-selectin, and IL-1β in male rats with induced periodontitis showed that H. perforatum had anti-inflammatory properties and significantly decreased all the inflammatory parameters [29]. Our results also showed lower inflammation in the group treated with H. perforatum compared to the normal saline solution group.
NF-κB plays a key role in the regulation of many genes that are responsible for the generation of mediators or proteins in inflammation, including TNF-α, IL-1β, and iNOS. Verma et al. [30] demonstrated that the H. perforatum treatment inhibited degradation of IκB-α and significantly decreased translocation of NF-κB. We clearly confirmed a significant increase in the production of IL-1β at 10-day interval after ligation.
Damlar et al. [31] showed that H. perforatum oil extract improved bone defects that were filled with bovine xenografts. We also had less bone loss in the H. perforatum group, consistent with the results of previous studies.
Osteoblasts and osteoclasts regulate bone turnover and are involved in bone formation and resorption, respectively. It had been demonstrated that H. perforatum extract increases MG-63 human osteoblast cell proliferation and increases bone formation by stimulating osteoblast differentiation and proliferation. It also reduces bone resorption by up regulating gene expression of osteoprotegerin (OPG) which plays an important role in bone turnover [32].
Tanideh et al. [15] showed that both topical and systemic forms of H. perforatum had significant and positive effects on mucositis. Wound-healing activity of H.
perforatum extract seems to be mainly due to an increase in the stimulation of production of collagen by fibroblasts and the activation of fibroblast cells in polygonal shape, with a role in wound repair by closing the damaged area [33].
However, antibiotics are widely used for treating periodontitis, and their short-term effect is obvious.
Nonetheless, in recent years, the resistance of pathogenic microorganisms to antibiotics has been increased due

Conclusion
According to the results of the present study, mixed of C. officinalis and H. perforatum plants might be employed as alternative medicine for periodontitis.